The present invention relates to a process for expression of protein products in Aspergillus oryzae, recombinant DNA vectors, a promoter for Aspergillus and transformed fungi.
In the past, numerous processes have been developed for the production of polypeptides or proteins by means of the recombinant DNA technology. The main interest has been concentrated on bacteria and yeast, e.g. E. coli, Bacillus subtilis and Saccharomyces cerevisiae being well characterized species as regards for instance expression and selection systems.
Beside the above mentioned microorganisms, filamentous fungi, such as Aspergillus niger, are attractive candidates as host microorganisms for recombinant DNA vectors being well-characterized and widely used microorganisms for the commercial production of enzymes. Efforts have especially been concentrated on the development of transformation systems by which a selection marker permitting selection of transformants from the untransformed host microorganisms is used.
In the last few years different selection markers for the transformation of Aspergillus nidulans have been described and procedures have recently been developed for integrative transformation of the filamentous fungus Aspergillus nidulans for the purpose of investigation of the genetic and molecular processes controlling fungal cell differentiation.
Transformation of A. nidulans has been demonstrated by using plasmids containing the Neurospora crassa pry-4 gene (Ballance, D. J. et al., Biochem.Biophys.Res.Commun., 112 (1983) 284-289), the A. nidulans amdS gene (Tilburn, J. G. et al., Gene 26 (1983) 205-221), the A. nidulans trpC gene (Yelton, M. M. et al., Proc.Natl.Acad.Sci. U.S.A., 81 (1984) 1470-1474) and the A. nidulans argB gene (John, M. A. and Peberdy J., Microb.Technol. 6 (1984) 386-389). The transforming DNA was found to be integrated into the host genome at rather low frequencies (typically <1000 transformants/μg of DNA).
Very recently transformation of Aspergillus niger with the amdS gene of A. nidulans was described (Kelly, J. M. and Hynes, M. J., EMBO Journal 4 (1985), 475-479) and amdS was shown to be a potential selection marker for use in transformation of Aspergillus niger that cannot grow strongly on acetamide as a sole nitrogen source. Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans has also been described recently (Buxton, F. P. et al., Gene 37 (1985), 207-214).
So far no methods have been developed for expression of foreign proteins in the filamentous fungi Aspergillus oryzae mainly due to insufficient knowledge of how to control gene expression in this fungus and due to the lack of suitable selectable genetic markers on cloning vectors and it is one aim of the present invention to develop such methods.
The present invention is furthermore related to a process for recombinant DNA production of Humicola lipases and a recombinant Humicola lipase.
Humicola lipases are obtainable from strains of thermophilic Humicola sp., including thermophilic Thermomyces sp., such as H. lanuginosa (Griffon and Maublanc) Bunce, H. stellata Bunce, H. grisea var. thermoidea, Cooney & Emerson, H. insolens, Cooney & Emerson, Thermomyces ibadanensis, Apinis & Eggins, H. hyalothermophila Moubasher, Mazen and Abdel-Hafez, H. grisea var. indica Subrahmanyam, H. brevis var. thermoidea, Subrahmanyam and Thirumalachar and H. brevispora Subrahmanyam and Thirumalachar.
H. lanuginosa has also been described under the synonyms Thermomyces lanuginosus Tsiklinsky, Sepedonium lanuginosum Griffon and Maublanc, Sepedonium thermaphilium cyclosporum and S. thermaphilum ovosporum Velich, Acremoniella sp. Rege, Acremoniella thermophila Curzi and Monotospora lanuginosa (Griffon and Maublanc) Mason.
Moreover, the species Scytalidium thermophilum (Cooney & Emerson) Austwich was by Hedger (1975, The ecology of thermophilic fungi in Indonesia. In Biodegradation et Humification. Rapport du 1er Colloque International—Nancy 1974 (ed. G. Kilbertius, O. Reisinger, A. Mourey & J. A. Cancela Da Fonseca), Sarreguemines: Pierron Editeur-57206) considered to belong to Humicola insolens. 
Production of a Humicola lanuginosa lipase is described in Japanese unexamined patent publication No. 48-62990, and in EP patent application No. 87307684.8. The latter also teaches use of this lipase in lipolytic detergent additives.
Due to the world wide use of enzyme additives in detergents and due to the fact that Humicola lipases have turned out to be superior to known detergent lipases both as regards detergency and stability, commercial interest in such lipases is high.
In the production of industrial enzymes yields have always been important for the profitability of the production process. The traditional way of improvement is to mutate the wild strain so as to obtain higher yielding mutants. By means of recombinant DNA technology a further possibility is to transform the gene for the desired product into a host microorganism capable of producing higher yields than the wild strain or with other favourable characteristics.
Accordingly, another aim of the present invention to develop a method for the production of Humicola lipases by recombinant DNA-technology.